OBJECTIVE To determine the roles of Sp family member binding sites in growth hormone variant gene transcription. RESULTS To determine the role ubiquitous Sp famila transcription factors may play in GH-V transcription, we conducted co-transfection experiments in which varied amounts of Sp1, Sp3 or both factors were introduced into cells to determine if the factors can directly activate transcription from the promoter. Sp1 treatment induced a modest increase in both basal and cAMP-stimulated transcription; Sp3 had no stimulatory effect on either basal or cAMP-induced activity, and may have been inhibitory at higher levels. There was no synergistic activity between the two factors. These results suggest that Sp./3 either plays no role in, or are not limiting for GH-V transcription. A key fact of this promoter is that Sp sites and adjacent elements are required for transcription. We reasoned that since essential elements for transcriptional activation were located within both the -140/-111 and -65/-42 regions, that there was cooperative activity between factors bound in these two regions. We wished to determine whether there was a strict requirement for these two elements, or whether either of the elements could substitute for the other in tandem and thus provide a possible enhancer for yeast 1-hybrid screening approaches. Constructs were prepared with -140/-105 or -75/-35 concatamers (multiple forward and reverse arrangements). The results of transient transfection experiments demonstrated that there was a synergistic effect of addition of 1 or two additional copies of the -140/-105 element, and that there was a requirement for the copies to be in the same orientation (i.e., a head to head arrangement was not functional whereas a head to tail arrangement showed synergistic activity over a single copy). The results thus demonstrate that 1) multiple copies of ea ch of the elements are necessary for activation, 2) specific spatial organization is necessary for cooperative activation, and 3) isolated elements are functional with a heterologous promoter. KEY WORDS placenta, gene transcription, placental growth hormone, transcription factors FUNDING NIH R01 HD26458